Charged-particle microscopy is a well-known and increasingly important technique for imaging microscopic objects, particularly in the form of electron microscopy. Historically, the basic genus of electron microscope has undergone evolution into a number of well-known apparatus species, such as the Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM), and Scanning Transmission Electron Microscope (STEM), and also into various sub-species, such as so-called “dual-beam” tools (e.g. a FIB-SEM), which additionally employ a “machining” Focused Ion Beam (FIB), allowing supportive activities such as ion-beam milling or Ion-Beam-Induced Deposition (IBID), for example.
More specifically, in a SEM, irradiation of a specimen by a scanning electron beam precipitates emanation of “auxiliary” radiation from the specimen, in the form of secondary electrons, backscattered electrons, X-rays and cathodoluminescence (infrared, visible and/or ultraviolet photons), for example; one or more components of this emanating radiation is/are then detected and used for image accumulation purposes. In a TEM, the electron beam used to irradiate the specimen is chosen to be of a high-enough energy to penetrate the specimen (which, to this end, will generally be thinner than in the case of a SEM specimen); the transmitted electrons emanating from the specimen can then be used to create an image. When such a TEM is operated in scanning mode (thus becoming a STEM), the image in question will be accumulated during a scanning motion of the irradiating electron beam. More information on some of the topics elucidated here can be found in the WIKIPEDIA entries “Electron Microscope,” “Scanning Electron Microscope,” “Transmission Electron Microscopy,” and “Scanning Transmission Electron Microscopy.”
As an alternative to the use of electrons as irradiating beam, charged particle microscopy can also be performed using other species of charged particle. In this respect, the phrase “charged particle” should be broadly interpreted as encompassing electrons, positive ions (e.g. Ga or He ions), negative ions, protons and positrons, for instance. As regards non-electron-based charged particle microscopy, some further information can, for example, be gleaned from references such as the WIKIPEDIA entries “Focused Ion Beam” and “Scanning Helium Ion Microscope,” Escovitz et al., “Scanning Transmission Ion Microscope with a Field Ion Source”, Proc. Nat. Acad. Sci. USA 72(5), pp 1826-1828 (1975), and Varentsov et al., “First Biological Images with High-Energy Proton Microscopy,” available from the PUBMED Database.
It should be noted that, in addition to imaging and performing (localized) surface modification (e.g. milling, etching, deposition, etc.), a charged particle microscope may also have other functionalities, such as performing spectroscopy, examining diffractograms, etc.
A Charged-Particle Microscope (CPM) generally comprises at least the following components: (i) a vacuum chamber, connected to one or more vacuum pumps and containing one or more ports (e.g. load locks) for moving (individual and/or groups of) specimens into and out of the vacuum chamber, (ii) particle-optical column comprising a radiation source, such as a Schottky electron source or ion gun, and an illuminator, which serves to manipulate a “raw” radiation beam from the source and perform upon it certain operations such as focusing, aberration mitigation, cropping (with an aperture), filtering, etc. It will generally comprise one or more (charged-particle) lenses, and may comprise other types of (particle-)optical component also. If desired, the illuminator can be provided with a deflector system that can be invoked to cause its exit beam to perform a scanning motion across the specimen being investigated. (iii) A specimen holder, on which a specimen under investigation can be held and positioned (e.g. tilted, rotated). If desired, this holder can be moved so as to effect scanning motion of the specimen w.r.t. the beam. In general, such a specimen holder will be connected to a positioning system. When designed to hold cryogenic specimens, the specimen holder will comprise means for maintaining said specimen at cryogenic temperatures, e.g. using an appropriately connected cryogen vat. (iv) A detector (for detecting radiation emanating from an irradiated specimen), which may be unitary or compound/distributed in nature, and which can take many different forms, depending on the radiation being detected. Examples include photodiodes, CMOS detectors, CCD detectors, photovoltaic cells, X-ray detectors (such as Silicon Drift Detectors and Si(Li) detectors), etc. In general, a CPM may comprise several different types of detector, selections of which can be invoked in different situations.
In the particular case of a dual-beam microscope, there will be (at least) two particle-optical columns, for producing two different species of charged particle. Commonly, an electron column (arranged vertically) will be used to image the specimen, and an ion column (arranged at an angle) will be used to (concurrently) modify (machine/process) the specimen, whereby the specimen holder can be positioned in multiple degrees of freedom so as to suitably “present” a surface of the specimen to the employed electron/ion beams.
In the case of a transmission-type microscope (such as a (S)TEM, for example), a CPM will specifically comprise an imaging system, which essentially takes charged particles that are transmitted through a specimen (plane) and directs (focuses) them onto analysis apparatus, such as a detection/imaging device, spectroscopic apparatus (such as an EELS device), etc. As with the illuminator referred to above, the imaging system may also perform other functions, such as aberration mitigation, cropping, filtering, etc., and it will generally comprise one or more charged-particle lenses and/or other types of particle-optical components.
As already mentioned, an example of an apparatus as set forth in the opening paragraph above is a FIB-SEM, and an important (but non-limiting) example of the use of such an apparatus is in the preparation of so-called TEM lamellae. As indicated above, TEM specimens need to be very thin, and they are generally prepared using highly specialized techniques. In one such technique, a focused ion beam (FIB) is used to cut/slice/extricate one or more lamella/lamellae from a bulk specimen, whereby, in general, electron-beam imaging is used to find/position a particular zone of interest on a specimen that is mounted to the specimen holder, the FIB is used to perform various incisions necessary to liberate a lamella from the (identified zone of the) specimen, and the lamella thus differentiated from the rest of the specimen is picked up/moved using a needle-like manipulator, attached to a positioning stage.
Lamella produced in this manner can then be removed from the FIB-SEM (with the aid of said manipulator), and studied in a (S)TEM, or in other analysis apparatus. For some general information on TEM lamella preparation, see, for example, Muehle et al. in “Microscopy: Science, Technology, Applications and Education,” pp. 1704-1716, 2010 (Formatex). For more information on the use of a FIB-SEM to prepare specimens for life sciences studies, see, for example, Rigort et al., “Cryo-focused-ion-beam Applications in Structural Biology,” available online from the PUBMED Database. Both of these publications are incorporated herein by reference.
The preparation of such TEM lamella is generally challenging, but is particularly challenging in the case of cryogenic specimens. Typical examples of cryogenic specimens include biological samples (such as cells, cell components, single-cellular organisms, etc.), which, by their very nature, typically need to be stored and studied in a body of aqueous liquid (such as water, electrolyte, cell fluid, blood plasma, etc.). Since an aqueous liquid introduced into a (quasi-)vacuum environment of a CPM will start to outgas/boil, the specimen (sample and aqueous liquid) is first frozen before being exposed to vacuum. Typically, so as to prevent damage to the sample caused by the formation of (sharp) ice crystals, such freezing is performed very rapidly, with the aim of achieving sample vitrification (solidification into an amorphous, glass-like phase) without significant ice crystallization; such vitrification can, for example, be achieved by rapidly plunging a specimen into a bath of cryogen, e.g. as set forth in U.S. Pat. No. 9,116,091 and European Patent 2 853 847 (with the same assignee as the current disclosure).
When a cold body is introduced into a vapor-containing (e.g. partially humid) environment, vapor in that environment will tend to condense on the cold body. If the body is sufficiently cold, the condensate in question will form as a layer of frozen/congealed solid, e.g. water ice. In a CPM, this is generally highly undesirable, since ice (or other condensate material) on a specimen surface will tend to absorb/scatter/deform a charged-particle beam directed onto that surface and crystalline ice on a biological sample surface may cause irreparable damage to that surface.
As a result, when cryogenic specimens are introduced into a CPM vacuum chamber from a load port (e.g. a load lock, access door to a storage space, etc.), the vacuum chamber must be subjected to a (fresh, continuing, or supplementary) lengthy pump-down so as to ensure that any vapor that is inadvertently co-introduced with the specimen is thoroughly removed from its environment; in this way, one seeks to ensure that, once surface modification of the specimen begins, there will be no build-up of condensate on the freshly modified surface. This is a time-consuming operation that can introduce a considerable throughput penalty in a process workflow.